Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii [Protein Structure and Folding]

September 29th, 2014 by Buchinger, E., Knudsen, D. H., Behrens, M. A., Pedersen, J. S., Aarstad, O. A., Tondervik, A., Valla, S., Skȷak–Braek, G., Wimmer, R., Aachmann, F. L.

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-Dmannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure being composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the Amodule. In this study we have determined the NMR structure of the three individual Rmodules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using Small angle X-ray Scattering (SAXS). Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and ITC. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. SAXS analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers show strong interaction between AlgE4R and both oligo-M and MG, while no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared to AlgE6.
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